Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow

Lee, Jerry S.H.; Panorchan, Porntula; Hale, Christopher M.; Khatau, Shyam B.; Kole, Thomas P.; Tseng, Yiider; Wirtz, Denis

doi:10.1242/jcs.02899

Cited by 101 publications

(98 citation statements)

References 55 publications

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“…The micromechanical properties of the cytoplasm of control and transfected cells were measured using ballistic intracellular nanorheology [28][29][30]. Briefly, 100-nm diameter polystyrene fluorescent nanoparticles were ballistically injected in the cytoplasm of control and transfected cells using a biolistic gun (Bio-Rad, Hercules, CA).…”

Section: Ballistic Intracellular Nanorheology (Bin)mentioning

confidence: 99%

“…We recently used Ballistic Intracellular Nanorheology (BIN), a high-throughput approach allowing the rigorous quantification of mechanical properties of living cells [29], to confirm the loss of mechanical stiffness of Lmna −/− MEFs [27]. Lmna −/− MEFs also display abnormal localization of several LINC complexes components [9,35].…”

Section: The Integrity Of Linc Complexes Is Essential To Cytoskeletalmentioning

confidence: 99%

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Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

Stewart-Hutchinson

Hale

Wirtz

et al. 2008

Experimental Cell Research

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The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.

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Section: Ballistic Intracellular Nanorheology (Bin)mentioning

confidence: 99%

Section: The Integrity Of Linc Complexes Is Essential To Cytoskeletalmentioning

confidence: 99%

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

Stewart-Hutchinson

Hale

Wirtz

et al. 2008

Experimental Cell Research

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256

295

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“…As already mentioned above, latrunculin B does not affect conventional stress fibers as quickly as it does the actin cap. Cells treatment with low doses of drugs inhibiting myosin II/ ROCK-based actomyosin contractility 10,11 affected phosho-MLC content in the actin dominated by the actin cap as opposed to conventional stress fibers, which are not connected to the nucleus, although much more work is required to directly test this hypothesis. Recent FRAP analysis indicate that LINC complex components are highly dynamic.…”

Section: The Actin Cap In Healthmentioning

confidence: 99%

The perinuclear actin cap in health and disease

Khatau

Kim

Hale

et al. 2010

Nucleus

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“…Atomic force microscopy (AFM) has revealed that VECs become more resistant to indentation by AFM tip 6-24 h after the application of a LSS of 20 dyn/cm 2 , with a transient asymmetry between upstream and downstream parts (11). More recently, particle tracking microrheology (PTM) has been used to investigate the temporal changes in viscoelastic shear moduli of cells in the plane of application of LSS over time courses of seconds (12) and minutes (13). These studies, however, do not address the adaptation of the in-plane microrheological properties of VECs to LSS applied over periods of hours (i.e., the time scale of morphological remodeling), nor the anisotropy of this adaptation, which correlates strongly with the direction of the applied LSS, as shown in this article.…”

mentioning

confidence: 99%

Anisotropic rheology and directional mechanotransduction in vascular endothelial cells

Álamo

Norwich²,

Li³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Adherent cells remodel their cytoskeleton, including its directionality, in response to directional mechanical stimuli with consequent redistribution of intracellular forces and modulation of cell function. We analyzed the temporal and spatial changes in magnitude and directionality of the cytoplasmic creep compliance (⌫) in confluent cultures of bovine aortic endothelial cells subjected to continuous laminar flow shear stresses. We extended particle tracking microrheology to determine at each point in the cytoplasm the principal directions along which ⌫ is maximal and minimal. Under static condition, the cells have polygonal shapes without specific alignment. Although ⌫ of each cell exhibits directionality with varying principal directions, ⌫ averaged over the whole cell population is isotropic. After continuous laminar flow shear stresses, all cells gradually elongate and the directions of maximal and minimal ⌫ become, respectively, parallel and perpendicular to flow direction. This mechanical alignment is accompanied by a transition of the cytoplasm to be more fluid-like along the flow direction and more solid-like along the perpendicular direction; at the same time ⌫ increases at the downstream part of the cells. The resulting directional anisotropy and spatial inhomogeneity of cytoplasmic rheology may play an important role in mechanotransduction in adherent cells by providing a means to sense the direction of mechanical stimuli.anisotropy ͉ microrheology ͉ shear stress B lood vessels are exposed to flow-induced shear stresses, which are borne primarily by vascular endothelial cells (VECs) (1). VECs perform functions such as regulation of permeability, the production, secretion, and metabolism of biochemical substances, and modulation of vascular smooth muscle cell contractility. Sustained application (hours) of laminar shear stresses (LSS) to cultured VECs induces cell elongation and alignment along the flow direction (2). The actin stress fibers thicken and gradually align with flow (3), the focal adhesions relocate primarily to the upstream part of the cell (4), and cell-cell junctions are transiently disrupted (5). The structural reorganization of cytoskeleton leads to changes in subcellular microrheology that can play an important role in mechanosensing and signaling by redistributing the external forces among intracellular subdomains (6, 7). Existing evidence suggests that changes in subcellular microrheology, including directionality and polarity, could provide a mechanism for cells to sense external forces and their direction, modulate intracellular signaling, and regulate gene expression and cell turnover (8).The realization that mechanical polarity may modulate cell function has conferred special significance to measuring the spatiotemporal adaptation of rheological properties of VECs to shear stresses. Sato et al. (9) determined the viscous and elastic resistances to micropipette aspiration of VECs after 24 h of directional LSS and provided the first quantitative evidence of the adaptation of VE...

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Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow

Abstract: SummaryBallistic intracellular nanorheology reveals ROCKhard cytoplasmic stiffening response to fluid flow

Cited by 101 publications

References 55 publications

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

The perinuclear actin cap in health and disease

Anisotropic rheology and directional mechanotransduction in vascular endothelial cells

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